The patent applications cited supra. disclose, inter alia, the characterization and preparation of various hormone and hormone-like receptors, including steroid, thyroid, and retinoid receptors such as those represented by the glucocorticoid, mineralocorticoid, thyroid, estrogen related and retinoid classes, and specifically, the glucocorticoid, estrogen, aldosterone, and retinoic acid receptors themselves. These specific receptors have been the subject of considerable research and form the particular bases for the inventions disclosed and claimed in those patent applications. Similarly, the extant parallel scientific literature has focused on the specific receptors listed above from among the classes of receptors that are known to exist.
Those applications, although cast in terms generic in sweep, exemplify the use of transfection (transformation) of host cells for operatively introducing the DNA useful for the intended purpose of the bioassay disclosed. Those applications describe the use of bacterial cells, such as E. coli, as well as mammalian cells, such as those of the COS line of monkey kidney cells. Those applications do not specifically disclose the use of a lytic or nonlytic system of infection using viral expression vectors.
The use of viral expression vectors has been reported in the art. See Darnell, Nature 297, 365 (1982), Thummel et al. J. Mol. Applied Gen. 1, 435 (1982) ; Chu et al., Nature 289, 378 (1981); Post et al., Mol. Cell Biol. 2, 233 (1982); Davidson et al., J. Virol. 61, 1226 (1987) and Mackett et al., PNAS 79, 7415 (1982). Thus, the literature provides generally the methods and means for producing operative viral expression vectors harboring DNA encoding desired polypeptide, transfecting (infecting) hosts and culturing same so as to produce novel cell lines that produce polypeptides that are heterologous to the endogenous polypeptides of that cell line, and means utilizing such systems for purposes of devising so-called recombinant systems that can be used for the production of heterologous polypeptides, and so forth. Further, Vennstrom et al, Nature 340, 242 (1989) used recombinant vaccinia virus to express thyroid hormone receptors and measured via DNA footprinting receptor-DNA binding by use of a restriction fragment containing DNA of the thyroid response element.
In the preparation of recombinant expression systems, an enviable advantage that such virally based systems provide is the generation of copious amounts of the heterologous polypeptide encoded by the viral expression vector. However, documentation will predict that, for example, adenovirus and other viruses retard the synthesis of endogenous proteins required for cell viability. See Beltz, et al, JMB 131, 353 (1979). Mechanistically, it is believed that while the virus may not interfere with the normal transcription process, it is believed that the resultant transcripts somehow have difficulty in being transported properly to the cytoplasm where they are translated into polypeptide. Secondly, the viral messenger RNA transcripts are preferentially translated (not solely because of stoichiometry), robbing the cell of energy needed to produce its own endogenous proteins.
The present invention was predicated on the possibility of harnessing the advantages of a viral expression vector based system for use in the bioassay procedures disclosed and claimed in the aforecited patent applications. It was assumed that such viral based expression systems would generate sufficient receptor, and consequently, marker molecules so as to extend laboratory success into commercial reality. However, the disadvantages of viral based expression systems enumerated above evoked the predictably unanswerable question of whether viral DNA would so disrupt the cells in which the bioassay was being utilized that the assay would not work, or not sufficiently, for commercial exploitation. Thus, until the present invention, it remained unpredictable in fact whether the use of viral based expression systems for bioassay procedures that measure the effect of materials for activity as receptor ligands, would result in bioassay systems having commercial viability.
It is thus an object of the present invention to base such ligand binding bioassays using recombinant host cells coinfected with viral based expression vectors harboring DNA operative for bioassay functionality.